T- and B-Cell Neogenesis Recovers Efficiently in Children with Acute Leukemia Given an Alpha-Beta T-Cell Depleted Haplo-HSCT Followed By Infusion of Donor T-Cells Genetically Modified with Inducible Caspase 9 Suicide Gene (BPX-501 cells)

Background

αβ T- and B-cell depleted HLA-haploidentical HSCT (αβ haplo-HSCT) is an alternative promptly available and virtually applicable to any children with malignant and non-malignant disorders lacking an HLA-matched donor or in need of an urgent allograft (Locatelli et al. 2017). Through this approach, recipients can benefit immediately after transplantation from the anti-leukemia effect mediated by donor natural killer (NK) and γδ T cells, which confers protection also against infections. Nonetheless, even with this type of HSCT, the recovery of adaptive immunity is suboptimal. We have developed a further refinement of the approach based on the post-transplant infusion of a titrated number of donor T cells transduced with the inducible suicide gene iC9 (BPX-501 cells). This strategy has the potential to accelerate the recovery of adaptive immunity and to restore an efficient T-cell mediated graft-versus-leukemia (GvL) effect without the risk of inducing uncontrollable graft-versus-host disease (GvHD). Since reconstitution of a naïve T- and B-cell repertoire plays a key role in the long-term ability to respond to a broad range of pathogens, as well as to tumor antigens, we quantified T-cell receptor and kappa chain-deleting recombination excision circles (TREC and KREC), which represent reliable surrogate of T and B-cell neogenesis respectively.

Study design and patients

We analyzed samples of 48 children (M/F: 26/22) with acute leukemia (31 ALL, 17 AML) given an ab T-cell and B-cell depleted haplo-HSCT after a myeloablative regimen followed by the infusion of a titrated number of donor BPX-501 cells (1 million/Kg) after a median of 27 days (range: 11-87). Median age at transplantation was 8.3 years (range, 0.9-18). Thirty-two and 16 patients did or did not receive a preparative regimen containing total body irradiation (TBI). Anti-T lymphocyte globulin (ATLG Grafalon®, Neovii Biotech, 12 mg/Kg) was administered from day -4 to -2 for preventing graft rejection and GvHD. Moreover, to reduce the risk of EBV-related PTLD, on day -1, patients received rituximab (200 mg/m2). We analyzed T-cell reconstitution by measuring the TREC, small DNA excision circles of the TCR d locus deleted during recombination of the a-locus and present in the majority of functional ab-T-cells (sjTREC), and during the TCR b-chain recombination (bTREC). We also analyzed B-cell reconstitution by measuring coding-joint (Cj), signal-joint KREC (sjKREC) which reflect newly produced naïve B cells and the mean number of B-cell division (n) using the formula n=LOG(Cj/sjKREC)/LOG2. We performed real-time quantitative PCR, as recently described (Arruda et al. 2018), on genomic DNA extracted from PBMC collected at 6 different time points (before and 1, 3, 6, 12 and 18 months after the allograft).

Results

Recovery of thymic function started at 3 months after transplant: sjTREC and bTREC amounts reached and overcame pre-transplant values at 6 months, continuing to increase until 18 months after HSCT. Number of B-cell divisions obtained by the measurement of cj and sjKREC found in peripheral blood matched with the newly-generated B-cells bone marrow output and showed the same kinetics of sj and bTREC, rising from the 3^rd month after HSCT. All these data correlate with immunophenotyping findings, which show a progressive increase over time of both total CD3+ and ab-T-cells. Furthermore, we observed a negative impact on both T- and B-cell neogenesis played by aGvHD (occurring in 14 patients), which became significant from 3 months after HSCT for sjTREC (p=0.013) and at 6 months for sjKREC (p= 0.03). We did not observe any impact of the use of TBI during the preparative regimen on T- and B-cells generation in this cohort.

Conclusions

BPX-501 cell infusion aims to contribute to the acceleration of immune reconstitution after a/b haplo-HSCT. In this study, we show that naïve T- and B- cells are quickly and efficiently generated in the early post-transplant period. This finding represents an interesting result considering the fully myeloablative conditioning regimens with the large use of TBI and the Ta/b and B-cells depletion (also in vivo by the administration of Rituximab) received by the patients. We observed an expected detrimental effect of aGvHD on immune reconstitution, but we emphasize the low frequency of this complication in this transplant setting despite the absence of any post-transplant pharmacological prophylaxis.